Microtubules and nucleoside diphosphate kinase. Comparison of kinetics of GTP- and CTP-induced assembly

The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.